The long-term objectives are to determine the mechanisms by which catecholamine (CA)-containing cells replenish endogenous CA stores lost via neuronal activity/secretion and, ultimately, the rules governing whether these mechanisms result in homeostasis vs a more or less lasting change in secretory output. Repletion of CA stores results from an increase in CA biosynthesis consequent to an increase in the catalytic activity of tyrosine hydroxylase (TH), the initial and rate-limiting enzyme. One mechanism by which CA cells increase TH activity is phosphorylation of the enzyme. In intact CA systems, TH is phosphorylated at four different residues, and the phosphorylation of three of these sites is regulated physiologically: Ser19 and Ser40 (phosphorylated by Ca/CaM- and cAMP-dependent protein kinase, respectively) and Ser31. This latter site and the protein kinase system responsible for its phosphorylation (ERKs, MAP kinases) represent a quite recently identified kinase/substrate classification, and little is known yet regarding their importance in regulating CA function. The specific objectives of the proposal are to (1.) characterize the regulation and relative involvement of different ERK species in Ser31 phosphorylation, (2.) establish the characteristics of site-specific TH phosphorylation in vitro using the appropriate, purified protein kinases, (3.) determine whether site-specific phosphorylation influences phosphorylation at other sites in vitro, (4.) evaluate and characterize the effects of site-specific phosphorylation and multiple-site phosphorylations upon the catalytic activity of TH in vitro, (5.) evaluate the interactions in vitro between CA-dependent and phosphorylation-dependent alterations in TH activity, and (6.) determine the effects of manipulating multiple-site TH phosphorylation in intact cells upon CA biosynthesis rates and upon the catalytic activity and CA binding characteristics of TH isolated from the cells. In terms of methods, (1.) Antisense oligonucleotides, to inhibit the expression of different ERK species in PC12 cells, will be tested for effects on Ser31 phosphorylation (relative to Ser8, Ser19 and Ser40) in intact PC12 cells. (2-3.) Kinetic parameters of site-specific TH phosphorylation (verified by HPLC of limit tryptic digests) will be studied in vitro using purified protein kinases (Ca/CaM-dependent, cAMP- dependent, ERK) with native PC12 TH and unphosphorylated recombinant wild type TH. Initial rates will be used to study interactions among sites. Mutant THs with A replacing Ser19, Ser31, or Ser40) will provide null/non-phosphorylatable controls while TH mutants with E as replacement will provide "phosphorylated" controls. (4-5.) The catalytic activity of TH (selected phosphorylation conditions/TH forms) will be measured in terms of cofactor/substrate kinetics and CA inhibition. A novel CA-TH interaction with recombinant TH will also be studied. (6.) Using previously established treatment profiles, specific patterns of multiple-site TH phosphorylation in situ will be tested for effects on CA biosynthesis rates and predicted effects upon catalytic activity and CA content of TH isolated from the cells.